Cardiovascular diseases account for more deaths than any other disease in western countries. A 2014 report indicates that the cost associated with cardiovascular health problems is now more than $310 billion per annum. Evidence suggests that the drug heparin can be an effective short-term treatment for many of the symptoms of cardiovascular disease, and laboratory studies indicate that heparin slows vascular smooth muscle cell growth, a component of late stage vascular disease. The long-term research goals in the PI's laboratory are to understand how heparin results in changes in vascular cell function at the molecular level. Such knowledge is likely to contribute to the development of advanced treatments for vascular diseases. Previously, the PI's laboratory developed antibodies that mimic heparin effects on vascular cells in culture and those antibodies have been used in studies that resulted in evidence for how vascular smooth muscle cell proliferation is inhibited in addition to helping the PI's laboratory identify a candidate heprin receptor. The research aims for the current proposal will enhance that understanding by first confirming the identification of the heparin receptor through knockdown strategies and by making targeted mutations in the likely heparin binding sequences. Second, the possible involvement of controlled activation of nitric oxide synthase by heparin will be investigated, and specific changes in gene expression for which the lab has preliminary evidence will be confirmed. Third, the PI's group will follow up on our previous evidence to clarify the anti-inflammatory effects of heparin on endothelial cells which also express the candidate heparin receptor noted above. If heparin treatment results in decreased inflammatory changes in gene expression and endothelial physiology, the data will support the hypothesis that heparin signaling acts counter to inflammatory signaling in the vasculature. In addition, information from these proposed studies will provide evidence of whether laminar flow and heparin result in additive anti-inflammatory responses, or work through the same pathways to achieve their responses. Fourth, the PI's lab group will develop an animal model to study these issues. Specifically, zebrafish (which express the candidate heparin receptor) will be investigated as a system in which genetics can be easily manipulated and in which heparin effects can then be studied. This animal model is very accessible to students, and will allow us to make significant progress in understanding the role of heparin in the vasculature. Coupled with the other results of this work, the zebrafish model will help provide a solid understanding of the molecular mechanisms by which heparin alters vascular cell behavior and suggestions for ways in which the heparin receptor could be a target for advanced therapies for vascular diseases.